The necessity and desirability of separating blood samples into their respective component parts, for example the cellular components and plasma, for test purposes and other medical applications has long been recognized. These applications are described in length in commonly assigned patent application Ser. No. 290,267 entitled "Particle Washing System and Method of Use", now U.S. Pat. No. 4,435,293, by Graham et al. That application is fully incorporated herein by reference.
The principles taught by Graham et al. are not limited to hematology type applications but may additonally be employed in immunoassay technology. Immunoassay technology is primarily directed towards the use of serologically specific reactions for the determination of the presence of specified antigens or antibodies, either of which may be called a determinant. Often these assays incorporate solid phase components wherein one of the serological determinants, i.e. either the antigen or the antibody, is coated upon a solid substrate such as a microsphere, red blood cell, or other type of microparticle. Following the serological reaction between the antigen and the specifically reactive antibody, the microparticles may be removed from the suspension along with the antigen antibody complex by centrifugation. Thus, unreacted serological determinants remain in solution and can be easily removed prior to identification and quantification of the extent of serological reaction and thus, in turn, quantification of the presence of the determinant to be identified.
The microparticles may take the form of microspheres which can be produced in various size ranges. The predominant size selected will depend upon the desired sensitivity and type of immunoassay performed. Often, in order to insure thorough mixing of the microparticles and other serological reagents, some form of vigorous agitation is required such as vortexing. It has been found that in these rare instances, the devices taught by Graham et al. employing reduced orifices are not always wholly successful in preventing leakage of the mother solution containing the sample and microparticle-immunoassay reagent through the orifice when mixing or when placed in contact with the outer wash solutions. It is an object of the present invention to provide an improved method for eliminating both premature leakage of the mother solution and unwanted mixing of the mother solution and wash solution in the embodiments of the Graham et al. invention employing capillary type orifices when used in operations requiring a high level of mechanical agitation.
Although there are many other types of separation devices available, typically, they are intented to facilitate recovery of the mother solution portion of the suspension and none is intended to permit the facile collection of microparticles from the mother solution or solve the aforementioned problem encountered in the Graham et al. embodiments employing a small diameter orifice.
For example, U.S. Pat. No. 3,932,277 to McDermott et al., directed to the separation of blood fractions, describes a system of tubes, one insertable into the other, whereby one tube inserts a barrier to separate the serum from the red blood cells after centrifuging in an attempt to prevent the mixing of the cells and the serum during decantation of the serum supernatant. During the insertion of the inner tube whereby the barrier is placed between the aforementioned portions, it is possible to have the serum filtered as it passes into the interior of the inner tube. Thus, this invention is directed towards the recovery of serum and requires great care in the placement of the barrier at the surface of the compacted red blood cell portion so as to avoid inadvertant mixing at that interface. Once in place, the barrier will prevent the removal of the red blood cells upon decantation of the serum. Thus, the barrier defeats a technician interested in working with the red blood cell layer from obtaining that cell layer. The McDermott et al. system additionally fails to provide apparatus or methods useful for immunoassays applications employing microparticles.
Similarly, U.S. Pat. No. 4,035,294 to Landers et al. is directed towards the collection, filtration and removal of the supernate following centrifugation. Landers et al. teach the insertion of an inner tube having a filter mounted at the bottom whereby, with the application of force to the inner tube upon insertion, the supernate is filtered through the membrane and is removably collected in the inner tube. As with the previously described reference, the disclosure of Landers et al. teaches an improved method in the filtration and handling of liquid supernate materials and fails to supply needed apparatus and methodology for a superior handling of separated microparticles from an immunoassay reaction solution, an object of the present invention.
U.S. Pat. No. 4,244,694 to Farina et al. describes the use of a reactor/separator device for use in automated solid phase immunoassays. The described device employs a water impermeable disc capable of supporting immunoabsorbents, immobilized antisera, ion exchange resins and other similar materials for reaction with reagents added to the inner tube upon centrifugation. Following the desired reaction, additional centrifugal forces are applied in order to force the aqueous phase through the filter making it water permeable thus permitting separation of desired components. Farina's invention provides a device wherein centrifugal force is employed for the mixing, transferance and separation of reactants in a reactor cavity separated from the collection chamber by a water impermeable disc. Such a device fails to solve the problems enumerated above, specifically those related to the collection and washing of particles suspended in a solution where a minimum of steps and a maximization of economy is desired.
Although the collection of microparticles from an immunoassay reaction solution has been described, it is to be understood that this is by way of illustration and that some of the conventional procedures described as well as the present invention are equally applicable to the separation of particles in general from a mother solution by application of gravitational forces.
It is an object of the present invention to permit the rapid separation and washing of microparticles from a solution in a "one step" operation. It is a further object that the present invention be capable of withstanding physical agitation during an immunoassay reaction without mixing of the reaction solution with the wash solutions. It is another object that during separation of the particles from the solution containing the particles, the particles are washed so as to remove any nonspecific serum coating and to dilute any solute drag. It is yet another object that the original particle containing sample solution be separately maintained from the sedimented particles to permit the facile removal of the original sample solution in order to reduce contamination. It is still another objective of the present invention that these objectives be accomplished in a simple system capable of economical production and employable within simple, inexpensive centrifuges commonly available. It is a further objective of the present invention to not only provide devices but also methodology capable of meeting the desired objectives. These and other objectives will readily become apparent to those skilled in the art in light of the teachings set forth herein.